The present invention pertains generally to immunoassays for determining the presence of a particular analyte, and in particular, to a test device which may be used to detect the presence of semen.
Immunoassay test devices are well known in the art. These devices are employed to detect a wide variety of substances. For example, U.S. Pat. No. 4,313,734 shows a method, test kit, and labeled component for the detection and/or determination of one or more components of the reaction between a specific binding protein and the corresponding bindable substance, in which one or more labeled components are used, that are obtained by coupling particles of a dispersion of a metal, metal compound or polymer nuclei, coated with a metal or metal compound, having a particle size of at least 5 nm, directly or indirectly to the desired component of the reaction. During the reaction or after an adequate reaction time, the physical properties and/or the amount of the metal and/or the formed metal containing agglomerate, is/are determined in the test sample, or optionally after a separation of the bound and free metal labeled components in one of the derived fractions.
U.S. Pat. No. 4,376,110 illustrates xe2x80x9ctwo-sitexe2x80x9d or xe2x80x9csandwichxe2x80x9d immunometric assay techniques for determination of the presence and/or concentration of antigenic substances in fluids using monoclonal antibodies. One monoclonal antibody is presented in a soluble labeled form and a second monoclonal antibody is presented bound to a solid carrier. The soluble and bound monoclonal antibodies may be the products of either the same or different cell lines. Each monoclonal antibody has an affinity for the antigenic substances of at least about 108 liters/mole.
U.S. Pat. No. 4,435,504 defines a chromatographic immunoassay employing a specific binding pair member and a label conjugate which delineate a border whose distance from one end of the chromatograph relates to the amount of analyte present. By combining the label conjugate and sample in a solution and immunochromatographing the solution, or employing a combination of enzymes, one enzyme being the label and the other enzyme affixed to the chromatographic support, the position of the border defined by the label can be related to the amount of analyte in the sample solution. Preferably, an immunochromatograph is employed having both a specific binding pair member and an enzyme affixed to the support. A sample is chromatographed and the amount of analyte is determined by (1) contacting the chromatograph with a second enzyme conjugated with a specific binding pair member which binds to the chromatograph in proportion to the amount of analyte bound to the chromatograph, or (2) including the second enzyme conjugate with the sample, resulting in a defined border related to the amount of analyte in the sample. The two enzymes are related in that the substrate of one is the product of the other, so that upon contact of the chromatograph with appropriate reagents, a detectable signal develops which permits detection of the border to which the analyte traveled. This distance can be related to the amount of analyte present in the sample.
U.S. Pat. No. 4,703,017 concerns a solid phase assay for an analyte where the binder is supported on a solid support, such as nitrocellulose, and the tracer is comprised of ligand labeled with a colored particulate label, such as a liposome including a dye. The assay has a high sensitivity, and the tracer is visible on the support under assay conditions without instrumentation and without further treatment.
U.S. Pat. No. 4,855,240 consists of a test device and assay for determining an analyte where the tracer and sample may be simultaneously applied to different absorbent material portions both in capillary flow communication with an absorbent material portion. The sample contacts the binder prior to any substantial contact between the sample and tracer or the tracer and binder.
U.S. Pat. No. 4,954,452 describes a method of performing a diagnostic immunoassay utilizing colloidal non-metal particles having conjugated to them a binding component capable of specifically recognizing an analyte to be determined. After reaction of the sample and colloidal non-metal particles, the presence or amount of analyte/colloidal non-metal particle complexes is determined by optical analysis as a measure of the amount of analyte in the sample. The method can be utilized for the specific detection of numerous analytes and is sensitive and has a wide detection range.
U.S. Pat. No. 5,028,535 is directed to a ligand-receptor assay for determining the presence or amount of at least one target ligand capable of competing with a ligand analogue conjugate for binding sites available on a ligand receptor. The ligand analogue conjugate has at least one ligand analogue coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing the target ligand. The assay includes the steps of: a. contacting the fluid sample with the ligand analogue conjugate and ligand receptor to form a reaction mixture, the relative amounts of ligand analogue conjugate and ligand receptor being such that in the absence of the target ligand, and subsequent to substantially equilibrium binding, substantially all of the ligand analogue conjugate is bound to the ligand receptor; b. detecting the unbound ligand analogue conjugate; and, c. relating the detectable signal to the presence or amount of target ligand in the fluid sample. In one embodiment, an optional means also is employed for removing the receptor from the reaction mixture. In other assay formats, the analyte of interest may be either a ligand receptor or ligand.
U.S. Pat. No. 5,075,078 shows an improved chromatographic strip binding assay device for determining the presence or amount of an analyte present in a patient sample. Assay label reagents interact with capture reagents immobilized in a testing region on the strip substrate to generate a visually detectable image indicative of the test result. The test result images include a minus sign (xe2x88x92) to indicate a negative test result if the suspect analyte is absent in the patient sample and a plus sign (+) to indicate a positive test result if the suspect analyte is present or is present at a pre-determined concentration in the patient sample. The immobilized capture reagents responsible for the location and configuration of the test result images are applied to the strip at an angled orientation with respect to the fluid flow direction of the strip to ensure that sharp, substantially complete test result images are formed during performance of the assay. The devices are designed to provide substantially self-performing assays having inherently clear test results which are not subject to misinterpretation by the skilled or untrained user.
PCT Application WO 95/16207 is directed to an assay device with a barrier for regulating reagent application. An assay device for detection and/or determination of an analyte in a test sample uses a barrier containing an aperture to control the application of reagents to the device for greater reproducibility of results.
U.K. Patent 2,204,398 pertains to an analytical test device for use in assays. The device is suitable for use in the home, clinic, or doctor""s surgery, and is intended to give an analytical result which is rapid and which requires the minimum degree of skill and involvement from the user. In a typical embodiment, the test device comprises a hollow casing containing a dry porous carrier which communicates directly or indirectly with the exterior of the casing such that a liquid test sample can be applied to the porous carrier.
In 1971 Hara et al. first described a protein in the seminal fluid, named gamma-seminoprotein. In 1978, Sensabaugh et al. characterized the protein in detail, found that its molecular weight corresponds to 30,000 Dalton, and named it p30. In 1980, Graves and Sensabaugh described the development of immunometric assays that demonstrated that p30 is a reliable forensic marker for the identification of semen. A range of 200,000 to 5.5 million nanograms of p30 per ml of semen was described. Other methods of detection of p30 have been developed including Ouchterlony double diffusion, crossover electrophoresis, rocket immunoelectrophoresis, radial immunodiffusion, and ELISA. A disadvantage of all these conventional methods is that they are either not sensitive enough or cumbersome and time consuming to perform in forensic laboratories.
The present invention is directed to a test device of the immunoassay variety which detects the presence of semen. The test device of the present invention overcomes the disadvantages of prior art semen tests, provides accurate results, and can be performed and results available in about 10 minutes or less. The test device can be used in hospitals, sexual assault centers, trauma centers, forensic laboratories, and at field sites. A preferred name is Abacus OneStep ABAcard(copyright) p30 Test. The test is sensitive to 4 ng of p30 per ml. Therefore, seminal fluid diluted up to 1 in a million is detectable.
Principle Behind the Test
In this test procedure, 200 xcexcl of sample is added to the sample well xe2x80x9cSxe2x80x9d, and allowed to soak in. If p30 is present in the semen specimen, it will react with the mobile monoclonal antihuman p30 antibody forming a mobile antigen-antibody complex. This mobile antibody-antigen complex migrates through the absorbent device towards the test area xe2x80x9cTxe2x80x9d. A monoclonal antihuman p30 antibody is present in the test area xe2x80x9cTxe2x80x9d. This immobilized antibody captures the above complex so that an antibody-antigen-antibody sandwich is formed. The conjugated pink dye particles concentrate in a narrow zone on the membrane. When the p30 concentration in the sample exceeds 4 ng/ml, the pink dye particles will form a pink colored band in the test area xe2x80x9cTxe2x80x9d indicating a positive test result. As an internal positive control, p30 antibody-dye conjugates cannot bind to the antibody in the test area xe2x80x9cTxe2x80x9d, but are captured by an immobilized anti immunoglobulin antibody present in the control area xe2x80x9cCxe2x80x9d forming a complex. The captured pink dye particles will thus form a band in the control area xe2x80x9cCxe2x80x9d indicating that the test has worked properly and proper procedures have been followed. Thus, the presence of two colored lines, one in the test area xe2x80x9cTxe2x80x9d and other in the control area xe2x80x9cCxe2x80x9d, indicates a positive result, while a line only in the control area xe2x80x9cCxe2x80x9d indicates a negative result (provided no xe2x80x9chigh dose hook effectxe2x80x9d).
In accordance with a preferred embodiment of the invention, the test device for detecting semen comprises a strip having a test sample introduction station, a test station, and a control station, the stations being disposed in spaced apart relationship along the strip. In a ready for use test device, the test sample introduction station includes labeled p30 antibodies, the test station includes immobilized p30 antibodies, and the control station includes immobilized polyclonal antibodies.
In accordance with another preferred embodiment, the test sample introduction station includes labeled monoclonal p30 antibodies, and the test station includes immobilized monoclonal p30 antibodies.
In accordance with another preferred embodiment, the test station also includes immobilized human IgM antibodies with immobilized human IgM monoclonal antibodies being preferred.
In accordance with a important aspect of the invention, the labeled p30 antibodies have a label selected from the group consisting of colloidal gold, colloidal silver, carbon, latex, dye, and enzyme.
Other features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention.